Metabolomic and Lipidomic Profiling of Bacillus Using Two-Dimensional Tandem Mass Spectrometry

Abstract

Lipidomic and metabolomic profiles of sporulated and vegetative Bacillus subtilis and Bacillus thuringiensis from irradiated lysates were recorded using a quadrupole ion trap mass spectrometer modified to perform two-dimensional tandem mass spectrometry (2D MS/MS). The 2D MS/MS data domains, acquired using a 1.2 s scan of negative ions generated by nanoelectrospray ionization of microwave irradiated spores, showed the presence of dipicolinic acid (DPA) as well as various lipids. Aside from microwave radiation to extract DPA and lipids from spores, sample preparation was minimal. Characteristic lipid and metabolic profiles were observed using 107108 cells of the two Bacillus species. Major features of the lipid profiles observed for the vegetative states included sets of phosphatidylglycerol (PG) lipids. Product ion spectra were extracted from the 2D MS/MS data, and they provided structural information on the fatty acid components of the PG lipids. The study demonstrates the flexibility, speed, and informative power of metabolomic and lipidomic fingerprinting for identifying the presence of spore-forming biological agents using 2D MS/MS as a rapid profiling screening method.

Machine-Learning Classification of Bacteria Using Two-Dimensional Tandem Mass Spectrometry

“…The 2D MS/MS spectra of the bacterial extract for each species show striking differences across the bacterial genus. In some cases, there are also differences between species of the same genus such as Bacillus subtilis (Figure A) and Bacillus thuringiensis (Figure B), in agreement with previous studies . Other organisms such as Staphylococcus aureus (Figure C) and Staphylococcus capitis (Figure D) are difficult to distinguish by their mass spectra alone.…”

“…In some cases, there are also differences between species of the same genus such as Bacillus subtilis (Figure 3 A) and Bacillus thuringiensis (Figure 3 B), in agreement with previous studies. 34 Other organisms such as Staphylococcus aureus (Figure 3 C) and Staphylococcus capitis (Figure 3 D) are difficult to distinguish by their mass spectra alone. Other organisms that are difficult to distinguish are Citrobacter freundii (Figure S3A) and Citrobacter koseri (Figure S3C).…”

“…This mass analysis technique has previously been used for (1) analysis of bacterial lipid dynamics as a function of growth time, (2) lipid and metabolomic profiling of Bacillus spp. spores, and (3) detection and structural characterization of sulfonamide antibiotics in mixtures . Another, and very different, 2D MS/MS experiment uses Fourier transform ion cyclotron MS (2D MS) .…”

Rapid Identification of Phenolics in Mixtures by Two‐Dimensional Tandem Mass Spectrometry with Microdroplet Accelerated Derivatization Reactions

“…2D MS/ MS has been used to analyze mixtures of fentanyls, chemical warfare agents, sulfonamides in milk, as well as lipids and metabolites in bacterial lysates. [1,5,[8][9][10] 2D MS/MS spectra display ion abundances for product ions with m/z values (Pro m/z) given on the y-axis and their corresponding precursor ions with m/z values (Pre m/z) given on the x-axis of the 2D plot (Figure 1B). All fragment ions resulting from collision-induced dissociation (CID) are represented by peaks that fall below the auto-correlation line where Pre m/z = Pro m/z.…”

“…The experiment is done using two frequency generators, the precursor scan taking about 1 second (for 1000 mass/charge units) while the product ion scans take just 1 ms to cover the same mass/charge range (Figure 1A). 2D MS/MS has been used to analyze mixtures of fentanyls, chemical warfare agents, sulfonamides in milk, as well as lipids and metabolites in bacterial lysates [1,5,8–10] …”